PROJECT SUMMARY Heterochromatin plays critical roles in maintaining genome stability and transcriptional gene silencing (TGS). It has become increasingly clear that misregulation of pathways influencing heterochromatin integrity cause or contribute to many human maladies, including numerous cancers. The composition and function of heterochromatin domains are largely conserved from the fission yeast Schizosaccharomyces pombe to humans and other metazoans. Heterochromatin establishment, epigenetic maintenance, and TGS in these organisms are complex processes regulated by numerous chromatin-associated factors. Despite such complexity, core principles of heterochromatin biology have been proposed but remain speculative. This study will experimentally test and articulate these core principles by distilling essential features of heterochromatin in a highly-controlled and orthogonal environment. This research proposal is composed of two aims. Aim 1 is to reconstitute histone 3 lysine 9 methylation (H3K9me)-dependent heterochromatin in Saccharomyces cerevisiae cells, which naturally lack H3K9me, with the goal of defining the minimal requirements for a repressive and heritable chromatin state conserved from fission yeast to human. Successful construction of an H3K9me-dependent heterochromatin domain in S. cerevisiae cells will provide a unique system for investigating how heterochromatin is epigenetically inherited and how it silences transcription and limits other DNA transactions. Accordingly, Aim 2 is to utilize in vivo reconstituted H3K9me-dependent heterochromatin to investigate the mechanism of TGS. H3K9me-dependent heterochromatin will be reconstituted in S. cerevisiae cells by sequentially recruiting S. pombe and human heterochromatin-associated proteins to a specific S. cerevisiae genomic locus. This minimal heterochromatin domain will then be utilized to test three models of TGS and assess the contribution of histone deacetylation and nucleosome remodeling to the formation of silent chromatin domains. A combination of synthetic biology-, next generation sequencing-, and proteomics-based experimental approaches will be utilized to execute this research plan. This work has potential to transform our mechanistic understanding of heterochromatin formation and thereby inform future studies aimed at reversing defects in heterochromatin-associated processes underlying human diseases. Furthermore, the reconstitution of heterologous heterochromatin domains in vivo with factors found in S. pombe and human cells will facilitate the development of cell-based assays amenable to high-throughput screens for small molecules that modulate the function of proteins and protein complexes involved in heterochromatin formation and disease progression. The proposed studies will thus deepen our understanding of fundamental processes underlying human diseases and open new avenues to their treatment.